The present invention relates generally to the cystic fibrosis (CF) gene, and, more particularly to the identification, isolation and cloning of the DNA sequence corresponding to the normal and mutant CF genes, as well as their transcripts and gene products. The present invention also relates to methods of screening for and detection of CF carriers, CF diagnosis, prenatal CF screening and diagnosis, and gene therapy utilizing recombinant technologies and drug therapy using the information derived from the DNA, protein, and the metabolic function of the cystic fibrosis transmembrane inductance regulator protein (CFTR).
CF is the most common severe autosomal recessive genetic disorder in the Caucasian population. It affects approximately 1 in 2000 live births in North America (Boat et al, The Metabolic Basis of Inherited Disease, 6th ed, pp 2649-2680, McGraw Hill, NY (1989)). Approximately 1 in 20 persons are carriers of the disease.
Although the disease was first described in the late 1930""s, the basic defect remains unknown. The major symptoms of cystic fibrosis include chronic pulmonary disease, pancreatic exocrine insufficiency, and elevated sweat electrolyte levels. The symptoms are consistent with cystic fibrosis being an exocrine disorder. Although recent advances have been made in the analysis of ion transport across the apical membrane of the epithelium of CF patient cells, it is not clear that the abnormal regulation of chloride channels represents the primary defect in the disease. Given the lack of understanding of the molecular mechanism of the disease, an alternative approach has therefore been taken in an attempt to understand the nature of the molecular defect through direct cloning of the responsible gene on the basis of its chromosomal location.
However, there is no clear phenotype that directs an approach to the exact nature of the genetic basis of the disease, or that allows for an identification of the cystic fibrosis gene. The nature of the CF defect in relation to the population genetics data has not been readily apparent. Both the prevalence of the disease and the clinical heterogeneity have been explained by several different mechanisms: high mutation rate, heterozygote advantage, genetic drift, multiple loci, and reproductive compensation.
Many of the hypotheses can not be tested due to the lack of knowledge of the basic defect. Therefore, alternative approaches to the determination and characterization of the CF gene have focussed on an attempt to identity the location of the gene by genetic analysis.
Linkage analysis of the CF gene to antigenic and protein markers was attempted in the 1950""s, but no positive results were obtained (Steinberg et al Am. J. Hum. Genet. 8: 162-176, (1956); Steinberg and Morton Am. J. Hum. Genet 8: 177-189, (1956); Goodchild et al J. Med. Genet. 7: 417-419, 1976).
More recently, it has become possible to use RFLP""s to facilitate linkage analysis. The first linkage of an RFLP marker to the CF gene was disclosed in 1985 (Tsui et al. Science 230: 1054-1057, 1985) in which linkage was found between the CF gene and an uncharacterized marker DOCRI-917. The association was found in an analysis of 39 families with affected CF children. This showed that although the chromosomal location had not been established, the location of the disease gene had been narrowed to about 1% of the human genome, or about 30 million nucleotide base pairs.
The chromosomal location of the D0CRI-917 probe was established using rodent-human hybrid cell lines containing different human chromosome complements. It was shown that DOCR1-917 (and therefore the CF gene) maps to human chromosome 7.
Further physical and genetic linkage studies were pursued in an attempt to pinpoint the location of the CF gene. Zengerling et al (Am. J. Hum. Genet. 40: 228-236 (1987) describe the use of human-mouse somatic cell hybrids to obtain a more detailed physical relationship between the CF gene and the markers known to be linked with it. This publication shows that the CF gene can be assigned to either the distal region of band q22 or the proximal region of band q31 on chromosome 7.
Rommens et al (Am. J. Hum. Genet. 43: 645-663, (1988) give a detailed discussion of the isolation of many new 7q31 probes. The approach outlined led to the isolation of two new probes, D7S122 and D7S340, which are close to each other. Pulsed field gel electrophoresis mapping indicates that these two RFLP markers are between two markers known to flank the CF gene, MET (White, R., Woodward S., Leppert M., et al. Nature 318: 382-384, (1985)) and D7S8 [Wainwright, B. J., Scambler, P. J., and J. Schmidtke, Nature 318: 384-385 (1985)), therefore in the CF gene region. The discovery of these markers provides a starting point for chromosome walking and jumping.
Estivill et al, (Nature 326: 840-845(1987)) disclose that a candidate cDNA gene was located and partially characterized. This however, does not teach the correct location of the CF gene. The reference discloses a candidate cDNA gene downstream of a CpG island, which are undermethylated GC nucleotide-rich regions upstream of many vertebrate genes. The chromosomal localization of the candidate locus is identified as the XV2C region. This region is described in European Patent Application 88303645.1. However, that actual region does not include the CF gene.
A major difficulty in identifying the CF gene has been the lack of cytologically detectable chromosome rearrangements or deletions, which greatly facilitated all previous successes in the cloning of human disease genes by knowledge of map position.
Such rearrangements and deletions could be observed cytologically and as a result, a physical location on a particular chromosome could be correlated with the particular disease. Further, this cytological location could be correlated with a molecular location based on known relationship between publicly available DNA probes and cytologically visible alterations in the chromosomes. Knowledge of the molecular location of the gene for a particular disease would allow cloning and sequencing of that gene by routine procedures, particularly when the gene product is known and cloning success can be confirmed by immunoassay of expression products of the cloned genes.
In contrast, neither the cytological location nor the gene product of the gene for cystic fibrosis was known in the prior art. With the recent identification of MET and D7S8, markers which flanked the CF gene but did not pinpoint its molecular location, the present inventors devised various novel gene cloning strategies to approach the CF gene in accordance with the present invention. The methods employed in these strategies include chromosome jumping from the flanking markers, cloning of DNA fragments from a defined physical region with the use of pulsed field gel electrophoresis, a combination of somatic cell hybrid and molecular cloning techniques designed to isolate DNA fragments from undermethylated CpG islands near CF, chromosome microdissection and cloning, and saturation cloning of a large number of DNA markers from the 7q31 region. By means of these novel strategies, the present inventors were able to identify the gene responsible for cystic fibrosis where the prior art was uncertain or, even in one case, wrong.
The application of these genetic and molecular cloning strategies has allowed the isolation and cDNA cloning of the cystic fibrosis gene on the basis of its chromosomal location, without the benefit of genomic rearrangements to point the way. The identification of the normal and mutant forms of the CF gene and gene products has allowed for the development of screening and diagnostic tests for CF utilizing nucleic acid probes and antibodies to the gene product. Through interaction with the defective gene product and the pathway in which this gene product is involved, therapy through normal gene product supplementation and gene manipulation and delivery are now made possible.
The gene involved in the cystic fibrosis disease process, hereinafter the xe2x80x9cCF genexe2x80x9d and its functional equivalents, has been identified, isolated and cDNA cloned, and its transcripts and gene products identified and sequenced. A three base pair deletion leading to the omission of a phenylalanine residue in the gene product has been determined to correspond to the mutations of the CF gene in approximately 70% of the patients affected with CF, with different mutations involved in most if not all the remaining cases.
With the identification and sequencing of the gene and its gene product, nucleic acid probes and antibodies raised to the gene product can be used in a variety of hybridization and immunological assays to screen for and detect the presence of either a normal or a defective CF gene or gene product. Assay kits for such screening and diagnosis can also be provided.
Patient therapy through supplementation with the normal gene product, whose production can be amplified using genetic and recombinant techniques, or its functional equivalent, is now also possible. Correction or modification of the defective gene product through drug treatment means is now possible. In addition, cystic fibrosis can be cured or controlled through gene therapy by correcting the gene defect in situ or using recombinant or other vehicles to deliver a DNA sequence capable of expression of the normal gene product to the cells of the patient.
According to an aspect of the invention, a DNA molecule comprises a DNA sequence selected from the group consisting of:
(a) DNA sequences which correspond to the DNA sequence as set forth in the following FIGS. 1A-1H from amino acid residue position 1 to position 1480;
(b) DNA sequences encoding normal CFTR polypeptide having the sequence according to the following FIGS. 1A-1H for amino acid residue positions from 1 to 1480;
(c) DNA sequences which correspond to a fragment of the sequence of the following FIGS. 1A-1H including at least 16 sequential nucleotides between amino acid residue positions 1 and 1480;
(d) DNA sequences which comprise at least 16 nucleotides and encode a fragment of the amino acid sequence of the following FIGS. 1A-1H; and
(e) DNA sequences encoding an epitope encoded by at least 18 sequential nucleotides in the sequence of the following FIGS. 1A-1H between amino acid residue positions 1 and 1480.
According to another aspect of the invention, a purified mutant CF gene comprises a DNA sequence encoding an amino acid sequence for a protein where the protein, when expressed in cells of the human body, is associated with altered cell function which correlates with the genetic disease cystic fibrosis.
According to another aspect of the invention, a purified RNA molecule comprises an RNA sequence corresponding to the above DNA sequence.
According to another aspect of the invention, a DNA molecule comprises a cDNA molecule corresponding to the above DNA sequence.
According to another aspect of the invention, a purified nucleic acid probe comprises a DNA or RNA nucleotide sequence corresponding to the above noted selected DNA sequences of groups (a) to (e).
According to another aspect of the invention, a DNA molecule comprises a DNA sequence encoding mutant CFTR polypeptide having the sequence according to the following FIGS. 1A-1H for amino acid residue positions 1 to 1480. The sequence is further characterized by a three base pair mutation which results in the deletion of phenylalanine from amino acid residue position 508.
According to another aspect of the invention, a DNA molecule comprises a cDNA molecule corresponding to the above DNA sequence.
According to another aspect of the invention, the cDNA molecule comprises a DNA sequence selected from the group consisting of:
(a) DNA sequences which correspond to the mutant DNA sequence and which encode, on expression, for mutant CFTR polypeptide;
(b) DNA sequences which correspond to a fragment of the mutant DNA sequences, including at least twenty nucleotides;
(c) DNA sequences which comprise at least twenty nucleotides and encode a fragment of the mutant CFTR protein amino acid sequence; and
(d) DNA sequences encoding an epitope encoded by at least eighteen sequential nucleotides in the mutant DNA sequence.
According to another aspect of the invention, purified RNA molecule comprising RNA sequence corresponds to the mutant DNA sequence.
A purified nucleic acid probe comprising a DNA or RNA nucleotide sequence corresponding to the mutant sequences as recited above.
According to another aspect of the invention, a recombinant cloning vector comprising the DNA sequences of the normal or mutant DNA and fragments thereof is provided. The vector, according to an aspect of this invention, is operatively linked to an expression control sequence in the recombinant DNA molecule so that the normal CFTR protein can be expressed, or alternatively with the other selected mutant DNA sequence the mutant CFTR polypeptide can be expressed. The expression control sequence is selected from the group consisting of sequences that control the expression of genes of prokaryotic or eukaryotic cells and their viruses and combinations thereof.
According to another aspect of the invention, a method for producing normal CFTR polypeptide comprises the steps of:
(a) culturing a host cell transtected with the recombinant vector for the normal DNA sequence in a medium and under conditions favorable for expression of the normal CFTR polypeptide; and
(b) isolating the expressed normal CFTR polypeptide.
According to another aspect of the invention, a method for producing a mutant CFTR polypeptide comprises the steps of:
(a) culturing a host cell transfected with the recombinant vector for the mutant DNA sequence in a medium and under conditions favorable for expression of the mutant CFTR polypeptide; and
(b) isolating the expressed mutant CFTR polypeptide.
According to another aspect of the invention, a purified protein of human cell membrane origin comprises an amino sequence encoded by the mutant DNA sequence where the protein, when present in human cell membrane, is associated with cell function which causes the genetic disease cystic fibrosis.
According to another aspect of the invention, the CFTR polypeptide is characterized by a molecular weight of about 170,000 daltons and an epithelial cell transmembrane ion conductance affecting activity.
According to another aspect of the invention, a substantially pure CFTR protein normally expressed in human epithelial cells and characterized by being capable of participating in regulation and in control of ion transport through epithelial cells by binding to epithelial cell membrane to modulate ion movement through channels formed in the epithelial cell membrane.
According to another aspect of the invention, a process for isolating the CFTR protein comprises:
(a) extracting peripheral proteins from membranes of epithelial cells to provide membrane material having integral proteins including said CFTR protein;
(b) solubilizing said integral proteins of said membrane material to form a solution of said integral proteins;
(c) separating said CFTR protein to remove any remaining other proteins of mammalian origin.
According to another aspect of the invention, a method is provided for screening a subject to determine if the subject is a CF carrier or a CF patient comprising the steps of providing a biological sample of the subject to be screened and providing an assay for detecting in the biological sample, the presence of at least a member from the group consisting of the normal CF gene, normal CF gene products, a mutant CF gene, mutant CF gene products and mixtures thereof.
According to another aspect of the invention, an immunologically active anti-CFTR polyclonal or monoclonal antibody specific for CFTR polypeptide is provided.
According to another aspect of the invention, a kit for assaying for the presence of a CF gene by immunoassay techniques comprises:
(a) an antibody which specifically binds to a gene product of the CF gene;
(b) reagent means for detecting the binding of the antibody to the gene product; and
(c) the antibody and reagent means each being present in amounts effective to perform the immunoassay.
According to another aspect of the invention, a kit for assaying for the presence of a CF gene by hybridization technique comprises:
(a) an oligonucleotide probe which specifically binds to the CF gene;
(b) reagent means for detecting the hybridization of the oligonucleotide probe to the CF gene; and
(c) the probe and reagent means each being present in amounts effective to perform the hybridization assay.
According to another aspect of the invention, a method is provided for treatment for cystic fibrosis in a patient. The treatment comprises the step of administering to the patient a therapeutically effective amount of the normal CFTM protein.
According to another aspect of the invention, a method of gene therapy for cystic fibrosis comprises the step of delivery of a DNA molecule which includes a sequence corresponding to the normal DNA sequence encoding for normal CFTR protein.
According to another aspect of the invention, an animal comprises an heterologous cell system. The cell system includes a recombinant cloning vector which includes the recombinant DNA sequence corresponding to the mutant DNA sequence which induces cystic fibrosis symptoms in the animal.
According to another aspect of the invention, a transgenic mouse exhibits cystic fibrosis symptoms.